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Claudia A Bertuccio, Tony T Wang, Kirk L Hamilton, Diego J Rodriguez-Gil, Steven B Condliffe, Daniel C Devor
We previously demonstrated endocytosis of KCa2.3 is caveolin-1-, dynamin II- and Rab5-dependent. KCa2.3 then enters Rab35/EPI64C- and RME-1-containing recycling endosomes and is returned to the plasma membrane (PM). Herein, we report on the mechanism by which KCa2.3 is inserted into the PM during recycling and following exit from the Golgi. We demonstrate KCa2.3 colocalizes with SNAP-23 and Syntaxin-4 in the PM of HEK and endothelial cells by confocal immunofluorescence microscopy. We further show KCa2.3 can be co-immunoprecipitated with SNAP-23 and Syntaxin-4...
2018: PloS One
Isabel M Schopp, Julien Béthune
To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated...
April 20, 2018: Journal of Visualized Experiments: JoVE
Shaojun Long, Kevin M Brown, L David Sibley
Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii . When combined with CRISPR/Cas9 based tagging, this method provides a robust approach to defining protein networks. This approach detects interaction within intact cells, it is applicable to both soluble and insoluble components, including large proteins complexes that interact with the cytoskeleton and unique microtubule organizing center that comprises the apical complex in apicomplexan parasites...
March 20, 2018: Bio-protocol
Ernesto Perez-Rueda, Rafael Hernandez-Guerrero, Mario Alberto Martinez-Nuñez, Dagoberto Armenta-Medina, Israel Sanchez, J Antonio Ibarra
Gene regulation at the transcriptional level is a central process in all organisms, and DNA-binding transcription factors, known as TFs, play a fundamental role. This class of proteins usually binds at specific DNA sequences, activating or repressing gene expression. In general, TFs are composed of two domains: the DNA-binding domain (DBD) and an extra domain, which in this work we have named "companion domain" (CD). This latter could be involved in one or more functions such as ligand binding, protein-protein interactions or even with enzymatic activity...
2018: PloS One
Chao Nie, Huimin Wang, Rui Wang, David Ginsburg, Xiao-Wei Chen
The flow of cargo vesicles along the secretory pathway requires concerted action among various regulators. The COPII complex, assembled by the activated SAR1 GTPases on the surface of the endoplasmic reticulum, orchestrates protein interactions to package cargos and generate transport vesicles en route to the Golgi. The dynamic nature of COPII, however, hinders analysis with conventional biochemical assays. Here we apply proximity-dependent biotinylation labeling to capture the dynamics of COPII transport in cells...
April 3, 2018: Proceedings of the National Academy of Sciences of the United States of America
Akshay Bareja, Conrad P Hodgkinson, Erik Soderblom, Greg Waitt, Victor J Dzau
The insulin-like growth factor 1 receptor (IGF1R) is a receptor tyrosine kinase with critical roles in various biological processes. Recent results from clinical trials targeting IGF1R indicate that IGF1R signaling pathways are more complex than previously thought. Moreover, it has become increasingly clear that the function of many proteins can be understood only in the context of a network of interactions. To that end, we sought to profile IGF1R-protein interactions with the proximity-labeling technique BioID...
April 27, 2018: Journal of Biological Chemistry
Teresa M Evans, Lindsay Bira, Jazmin Beltran Gastelum, L Todd Weiss, Nathan L Vanderford
No abstract text is available yet for this article.
March 6, 2018: Nature Biotechnology
Jiulia Satiaputra, Bart A Eijkelkamp, Christopher A McDevitt, Keith E Shearwin, Grant W Booker, Steven W Polyak
Biotin (Vitamin B7) is a critical enzyme co-factor in metabolic pathways important for bacterial survival. Biotin is obtained either from the environment or by de novo synthesis, with some bacteria capable of both. In certain species, the bifunctional protein BirA plays a key role in biotin homeostasis as it regulates expression of biotin biosynthetic enzymes in response to biotin demand and supply. Here, we compare the effect of biotin on the growth of two bacteria that possess a bifunctional BirA, namely Escherichia coli and Staphylococcus aureus...
April 2018: Applied Microbiology and Biotechnology
Cilly Bernardette Schnider, Damaris Bausch-Fluck, Francis Brühlmann, Volker T Heussler, Paul-Christian Burda
During their development within the vertebrate host, Plasmodium parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; however, only a limited number of proteins of this membrane have been identified so far. This is partially because systematic proteomic analysis of the protein content of the PVM has been difficult in the past, due to difficulties encountered in attempts to separate the PVM from other membranes such as the parasite plasma membrane...
January 2018: MSphere
Muthukumar Ramanathan, Karim Majzoub, Deepti S Rao, Poornima H Neela, Brian J Zarnegar, Smarajit Mondal, Julien G Roth, Hui Gai, Joanna R Kovalski, Zurab Siprashvili, Theo D Palmer, Jan E Carette, Paul A Khavari
RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA...
March 2018: Nature Methods
Kim Schilders, Evelien Eenjes, Gabriëla Edel, Anne Boerema de Munck, Marjon Buscop van Kempen, Jeroen Demmers, René Wijnen, Dick Tibboel, Robbert J Rottier
Sox2 is a Sry-box containing family member of related transcription factors sharing homology in their DNA binding domain. Sox2 is important during different stages of development, and previously we showed that Sox2 plays an important role in branching morphogenesis and epithelial cell differentiation in lung development. The transcriptional activity of Sox2 depends on its interaction with other proteins, leading to 'complex-specific' DNA binding and transcriptional regulation. In this study, we generated a mouse model containing a biotinylatable-tag targeted at the translational start site of the endogenous Sox2 gene (bioSox2)...
February 2018: Transgenic Research
Vaishali Verma, Charanpreet Kaur, Payal Grover, Amita Gupta, Vijay K Chaudhary
The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro...
2018: PloS One
Chenlu He, Gregory Custer, Jingheng Wang, Silvina Matysiak, Dorothy Beckett
Small molecules regulate transcription in both eukaryotes and prokaryotes by either enhancing or repressing assembly of transcription regulatory complexes. For allosteric transcription repressors, superrepressor mutants can exhibit increased sensitivity to small molecule corepressors. However, because many transcription regulatory complexes assemble in multiple steps, the superrepressor phenotype can reflect changes in any or all of the individual assembly steps. Escherichia coli biotin operon repression complex assembly, which responds to input biotin concentration, occurs via three coupled equilibria, including corepressor binding, holorepressor dimerization, and binding of the dimer to DNA...
February 20, 2018: Biochemistry
Julissa M Churata-Salcedo, Lucia M Almeida, Guillermo González, Robert D Gordon
The subgenus Sidonis Mulsant, 1850 is elevated to generic status and two new species from Brazil are described and illustrated: Sidonis bira sp. nov. and Sidonis biguttata sp. nov. New geographic distribution records are provided. In addition, lectotypes of Sidonis consanguinea (Mulsant, 1850) and S. guttata (Sicard, 1912) are designated. Illustrations of diagnostic characters from five of six species of the genus, comments on the differences from similar species and a key to all recognized taxa are included...
November 20, 2017: Zootaxa
Huijun Xue, Yuanyuan Bei, Zhengyan Zhan, Xiuqiang Chen, Xin Xu, Yu V Fu
Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots) have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios...
2017: Frontiers in Microbiology
Alexis Gabriel Rozenbaum, Dotan Shaked Gelband, Mordechai Stein, Henk K Mienis, Rivka Rabinovich
Despite the extensive geological and paleontological searches in the south Levant, no terrestrial fauna of late Neogene age was yet reported. Here, we report the first evidence of "ancient deer"-cervid in the late Miocene (Tortonian) lacustrine section of the Bira Formation at Hagal Stream, Jordan Valley, northern Israel. The section comprises rich assemblage of macrofauna fossils, mostly freshwater mollusks. The mammalian bone was discovered among the macrofauna fossils, and is described as an almost complete left humerus of an adult animal identified as an artiodactyls element probably of a cervid...
2017: PloS One
Jonas Lohse, Alexandra Schindl, Natasha Danda, Chris P Williams, Karl Kramer, Bernhard Kuster, Martin D Witte, Guillaume Médard
A method for identifying probe modification of proteins via tandem mass spectrometry was developed. Azide bearing molecules are immobilized on functionalised sepharose beads via copper catalysed Huisgen-type click chemistry and selectively released under acidic conditions by chemical cleavage of the triazene linkage. We applied this method to identify the modification site of targeted-diazotransfer on BirA.
October 31, 2017: Chemical Communications: Chem Comm
Nadine Opitz, Kerstin Schmitt, Verena Hofer-Pretz, Bettina Neumann, Heike Krebber, Gerhard H Braus, Oliver Valerius
The Asc1 protein of Saccharomyces cerevisiae is a scaffold protein at the head region of ribosomal 40S that links mRNA translation to cellular signaling. In this study, proteins that colocalize with Asc1p were identified with proximity-dependent Bio tin ID entification (BioID), an in vivo labeling technique described here for the first time for yeast. Biotinylated Asc1p-birA*-proximal proteins were identified and quantitatively verified against controls applying SILAC and mass spectrometry. The mRNA-binding proteins Sro9p and Gis2p appeared together with Scp160p, each providing ribosomes with nuclear transcripts...
December 2017: Molecular & Cellular Proteomics: MCP
Mauricio A Reynoso, Germain C Pauluzzi, Kaisa Kajala, Sean Cabanlit, Joel Velasco, Jérémie Bazin, Roger Deal, Neelima R Sinha, Siobhan M Brady, Julia Bailey-Serres
Isolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here, we describe transfer of the isolation of nuclei from tagged specific cell types (INTACT) to the monocot rice ( Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear-envelope-targeting domain of the nuclear tagging fusion (NTF) protein with an outer nuclear-envelope-anchored domain. This modified NTF was combined with codon-optimized Escherichia coli BirA in a single T-DNA construct...
January 2018: Plant Physiology
Thomas E H Bond, Alanna E Sorenson, Patrick M Schaeffer
Biotin protein ligase (BirA) has been identified as an emerging drug target in Mycobacterium tuberculosis due to its essential metabolic role. Indeed, it is the only enzyme capable of covalently attaching biotin onto the biotin carboxyl carrier protein subunit of the acetyl-CoA carboxylase. Despite recent interest in this protein, there is still a gap in cost-effective high-throughput screening assays for rapid identification of mycobacterial BirA-targeting inhibitors. We present for the first time the cloning, expression, purification of mycobacterial GFP-tagged BirA and its application for the development of a high-throughput assay building on the principle of differential scanning fluorimetry of GFP-tagged proteins...
December 2017: Microbiological Research
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